Chronic lymphocytic leukemia (CLL), an incurable malignancy of mature B-lymphocytes involves blood, bone marrow, and secondary lymphoid organs. A role of the tissue microenvironment in the pathogenesis of CLL is hypothesized based on in vitro observations but its contribution in vivo remain ill-defined. To elucidate effects of tumor host interactions in vivo we purified tumor cells from 24 treatment nave patients. Samples were obtained concurrently from blood, bone marrow and/or lymph node and analyzed by gene expression profiling. We identified the lymph node as a key site in CLL pathogenesis. CLL cells in the lymph node showed upregulation of gene signatures indicating B-cell receptor (BCR) and NF-&#61547;B activation. Consistent with antigen dependent BCR signaling and canonical NF&#61547;B activation, we detected phosphorylation of SYK and IkBalpha, respectively. Expression of BCR target genes was stronger in clinically more aggressive CLL indicating more effective BCR signaling in this subtype in vivo. Tumor proliferation quantified by expression of E2F and c-MYC target genes and verified by Ki67 staining by flow cytometry was highest in lymph node and correlated with clinical disease progression. To allow modeling of pathogenic tumor-host interactions we have established a mouse model that will be instrumental in testing specific interventions. In a second study we have focused on the role of CD44 a receptor for hyaluronic acid. We showed that CD44 engagement protects CLL cells from spontaneous and fludarabine-induced apoptosis. The anti-apoptotic effect appears to be mediated through activation of the PI3K/Akt and MAPK/ERK pathways and increased MCL-1 protein levels. PI3K or MEK inhibitors as well as obatoclax, an antagonist of MCL-1, blocked the pro-survival effect of CD44. Furthermore, obatoclax sensitized CLL cells to fludarabine resulted in a synergistic drug effect. Our findings emphasize the therapeutic potential of PI3K/AKT or MAPK/ERK inhibitors and obatoclax for combination chemotherapy approaches that overcome the supportive effect of the tissue microenvironment on CLL cell survival and drug resistance. We have investigated the potential of ON 01910.Na (Onconova, Therapeutics) a novel non-ATP competitive compound that can inhibit the PI-3K/Akt/mTOR pathway. We found that ON 01910.Na induced apoptosis of the leukemic cells in all CLL samples tested, without affecting T-cell viability. ON 01910.Na was equal cytotoxic against CLL samples showing adverse biologic and cytogenetic features. To further delineate the biological processes underlying ON 01910.Na induced apoptosis, we performed Gene Expression Profiling (GEP) in CLL cells treated in vitro These studies identified ON 01910.Na as a promising agent in the treatment of CLL with an interesting dual mechanism of action: activation of apoptotic stress signals leading to Noxa and BIM up-regulation, combined with inhibition of the BCR/PI3K/AKT pathway that can block microenvironment-induced survival and proliferation signals. Based on these data we initiated a phase I clinical trial of ON 01910.Na. Our findings that tumor proliferation is a result of tumor host interactions in vivo and a key determinant of clinical outcome can redirect therapeutic efforts. First, inhibiting the trafficking of CLL cells to protective microenvironment niches may be able to deprive the tumor cells of essential signals and induce apoptosis or sensitize the tumor to the effect of cytotoxic therapy. Furthermore, targeting essential signaling pathways in the microenvironment with small molecules could inhibit proliferation of CLL cells and thereby not only shrink tumor burden but also reduce clonal evolution. We identify the BCR as a valid therapeutic target in vivo, and small molecules inhibiting BCR signaling indeed show promise in early clinical trials. Another implication of our study is that tumor biology differs between anatomic locations possibly affecting drug sensitivity. For example we found increased expression of BCL2A1, an NF-&#954;B regulated gene in LN-resident cells. BCL2A1 is an anti-apoptotic member of the BCL-2 family of proteins that can confer resistance to BCL-2 inhibitors. Thus, the variable biology of CLL cells in the microenvironment presents therapeutic opportunities and challenges that we will aim to address in clinical trials with strong translational research components